Trace Element Solution

ABSTRACT

The inventions discloses a trace element solution, which comprises at least the following metals: zinc; manganese; selenium; and copper; and which comprises Vitamin B12. The solution furthermore comprises butaphosphan to stabilize the Vitamin B12 and the inclusion of butaphosphan may have synergistic activity with the minerals.

FIELD OF INVENTION

The present invention relates to a trace element solution.

More particularly, the present invention relates to a trace elementsolution supplemented with Vitamin B12.

BACKGROUND TO INVENTION

It has been found that there is a deficiency of certain trace elementsin pastures for livestock in particular areas around the world. Varioussuggestions have been made to provide the required trace elements tosuch animals. Different chemical compounds and complexes have beeninvestigated for applying the trace elements by way of licks, drenchesor injections.

In general the problem with injectable solutions is that theconcentrations of the minerals in the solutions is too low. This meansthat relatively large quantities have to be injected, which in turncauses tissue damage and also abscesses at the site of injection.Furthermore, it is generally the case that different trace elementsseldomly are individually sufficient. This means that two or more traceelement solutions have to be provided by way of separate injections.

ZA 1982/6778 (Laurie) discloses a trace element solution and a method ofproviding the trace elements to livestock. These trace element solutionsinclude ethylene diamino tetra acetic acid complex of the requiredmineral in suitable quantities. However, the trace element solutionincludes no selenium or selenite compound.

In the specification and claims the expression EDTA refers to ethylenediaminotetraacetic acid (C₁₀H₁₆O₈N₂ or (HO₂CH₂C)₂NCH₂CH₂N—(CH₂CO₂H)₂).

U.S. Pat. No. 4,335,116 (Howard) discloses mineral-containingtherapeutic compositions containing EDTA complexes of trace elements.Notably, U.S. Pat. No. 4,335,116 utilises tetra-sodium EDTA, a seleniumglycine complex, and metal chlorides for the preparation of the EDTAcomplexes. Unfortunately, the chloride ions cause contamination and eachcomplex solution is to be made individually. Furthermore, overnight timeis required for complexing and heating up afterward to speed up theprocess, requires extra apparatus. If mixtures are required, theindividual solutions are to be blended. If various concentrations aswell as compositions are to be made, it can only be done in a cumbersomeway, requiring extra apparatus. A further problem arises when mixturesof high concentration are needed. In certain cases it would beimpossible to deliver them, because mixing is always accompanied bydilution. The maximum concentration achieved with this method was 13.5mg/ml.

U.S. Pat. No. 6,638,539 (Laurie et al) discloses a method of preparing atrace element solution, which includes the steps of providing at leastone EDTA-complex, of providing a sodium selenite solution, and ofcombining the EDTA-complexes and the sodium selenite solution. However,the method enables production of a trace element solution of only about55 mg/ml.

U.S. Pat. No. 7,285,292 (Laurie et al) discloses a trace elementsolution, which comprises at least one metal selected from the groupcomprising selenium, copper, zinc, manganese and chromium and whichcomprises a concentration of the metal(s) of at least 60 mg/ml. Thesolution further comprises at least one compound selected from the groupcomprising iodine, potassium iodide, sodium iodide, iron, iron chloride,zinc oxide, manganese sulphate, sodium selenite, copper carbonate,sodium carbonate, anhydrous disodium EDTA and sodium hydroxide. Thetrace element solution is prepared by a method consisting essentially ofthe steps of preparing a MnCO₃ mixture in a container; adding anEDTA/NaOH mixture to the container and subsequently adding at least onemetal compound; and adding Na₂SeO₃ to the container to obtain the traceelement solution. The method also comprises the step of addingCrCl₃.6H₂O to the trace element solution.

Often various other nutritional, dietary or medical components need tobe added to the trace element solutions. Though which seems easy toachieve by merely mixing has not proven to be easy as the resultedmixture or solution becomes unstable and murky over time and hence thesolution needs to be discarded. The disadvantage is that the solutionscannot be stored for longer periods. Addition of further components ismore difficult than it appears resulting in both physical solutioninstability and in some cases such as vitamin B12 addition—chemicalinstability.

It is an object of the invention to suggest a trace element solution forovercoming these problems.

SUMMARY OF INVENTION

According to the invention, a trace element solution, comprises at leastthe following metals:

-   -   (a) zinc;    -   (b) manganese;    -   (c) selenium; and    -   (d) copper;        and comprises Vitamin B12.

The ratio of zinc to manganese may be at least 2:1.

The ratio of zinc to manganese may be at least 4:1.

The ratio of zinc to copper may be at least 2:1 or 4:1.

The ratio of zinc to selenium may be at least 4:1 or 12:1.

The concentration of the metals may be at least 36 mg/ml.

The trace element solution may comprise the following concentrations:

-   -   (a) at least 24 mg/ml zinc;    -   (b) at least 4 mg/ml manganese;    -   (c) at least 2 mg/ml selenium;    -   (d) at least 6 mg/ml copper; and    -   (e) at least 0.6 mg/ml Vitamin B12.

The concentration of the metals may be at least 90 mg/ml.

The solution may comprise chromium and/or iodine.

The solution may comprise butaphosphan to stabilize the Vitamin B12.

The inclusion of butaphosphan may enable the Vitamin B12 to remain morestable.

The inclusion of butaphosphan may have synergistic activity with theminerals.

The solution may be an injectable trace element solution.

The solution may be visually stable.

The invention also extends to a method of preparing a trace elementsolution as described herein.

DESCRIPTION OF EXAMPLE

The invention will now be described by way of an example of a traceelement solution in accordance with the invention.

The example relates to a trace element solution predominantly to be usedfor cattle and includes the mineral elements zinc, manganese, seleniumand copper and Vitamin B12.

According to the invention, a trace element solution, comprises thefollowing metals:

-   -   (a) zinc;    -   (b) manganese;    -   (c) selenium; and    -   (d) copper;        and comprises Vitamin B12.

The ratio of zinc to manganese can be at least 2:1.

The ratio of zinc to manganese can be at least 4:1.

The ratio of zinc to copper can be at least 2:1 or 4:1.

The ratio of zinc to selenium can be at least 4:1 or 12:1.

As an example the concentration of the metals is at least 36 mg/ml.

As an example the trace element solution comprises the followingconcentrations:

-   -   (a) at least 24 mg/ml zinc;    -   (b) at least 4 mg/ml manganese;    -   (c) at least 2 mg/ml selenium;    -   (d) at least 6 mg/ml copper; and    -   (e) at least 0.6 mg/ml Vitamin B12.

In a further example, the concentration of the metals can be at least 90mg/ml.

The solution can comprise chromium and/or iodine.

The solution comprises butaphosphan to stabilize the Vitamin B12.

The inclusion of butaphosphan may have synergistic activity with theminerals

The solution is generally an injectable trace element solution.

The solution is visually stable.

1. Objective of the Study

The objective of the study is to develop a new formulation of aninjectable trace element solution supplemented with Vitamin B12.

The new formulation is required to include the followingcharacteristics:

-   -   (a) Active ingredients: Cu (6 mg/ml), Mn (4 mg/ml), Zn (24        mg/ml) and Se (2 mg/ml), Vitamin B12 (0.6 mg/ml) (A further        example of the invention is to obtain a product with the        following formulation: Active ingredients: Cu (15 mg/ml), Mn (10        mg/ml), Zn (60 mg/ml) and Se (5 mg/ml), Vitamin B12 (1.5 mg/ml))    -   (b) Application: Mineral and vitamin supplement

2. Formulation Plan

The formulation plan consisted of performing two consecutive phases withthe aim to select at the end one of several formulation candidates.

-   -   First phase consisted of feasibility formulation study:

Based on known injectable processes, with organometallics complex withEDTA, incorporation of vitamin B12 was screened by testing 3 possibledifferent stabilizers.

-   -   Second Phase consists in evaluating the physical and chemical        stability of the formulations which have been developed during        precedent phase with the aim to select stable formulation        prototypes.

Physical stability study was performed at 5° C., 25° C./60% RH and 40°C./75% RH: T0, T1, T2 and T3 months. A visual inspection was checked atdifferent periods in order to control the absence of precipitation inthe formulations. Assay of vitamin B12 was followed up on stability at25° C./60% RH and 40° C./75% RH: T0, T3 and T6 months.

3. Formulation Studies 3.1. Phase 1: Feasibility Study

Based on known injectable processes, salts of zinc, copper and manganesewere dissolved in water and complexed with EDTA, and then salt ofselenium is added.

Incorporation of vitamin B12 with different stabilizers was screened.

Vitamin B12 is a water-soluble vitamin available as a dietarysupplement. Vitamin B12 exists in several forms. Vitamin B12cyanocobalamin is used for this study.

A vitamin B12 conforming to EP monograph was screened and received.

It is generally known that vitamins, and especially vitamin B12, are notvery stable in solution and degradation is observed on storage.

According bibliographic researches and trials, the following stabilizershave been tested:

-   -   (a) Butaphosphan    -   (b) Glycine    -   (c) Antioxidants

Butaphosphan (According Patent Application US2011/0065665A1)

Butaphosphan is a phosphonic derivative acid.

Butanol described in US2011/0065665A1 has been replaced by use of benzylalcohol (1%).

Note: butaphosphan is used with vitamin B12 in Catosal® formulation,from Bayer, and alone in Calphone® formulation, from Bayer.

Synonyms: Butaphosphan; (1-Butylamino-1-methyl-ethyl)-phosphinic acid

Molecular Formula: C7H18NO2P

Formula Weight: 179.20

CAS Registry Number: 17316-67-5

In US2011/0065665A1: 10% of butaphosphan, 0.005% of vitamin B12, 3.0% ofn-butanol/100% water Catosal® formulation: butaphosphan (100 mg),vitamin B12 (50 μg), n-butanol (30 mg)/1 ml Calphone®): formulation:butaphosphan (2 g)/500 ml

The following concentrations of butaphosphan have been tested:

-   -   5% w/v: a dark clear solution without particles is obtained    -   10% w/v: a dark clear solution without particles is obtained    -   20% w/v: incomplete solubilisation of butaphosphan

Then a quantity of stock solution of vitamin B12 was incorporated in themix to have a final concentration of 0.15% w/v.

Physical stability study was performed on 5 and 10% w/v butaphosphan(with vitamin B12) solutions at ambient temperature:

-   -   T1 week: dark clear solution    -   T2 weeks: dark clear solution    -   T4 weeks: dark clear solution

Glycine

Glycine is an aminoacid.

Glycine is used with vitamin B12 and selenium mineral in Biodyl®formula, from Merial. Biodyl® formulation: glycine (5.0 g), vitamin B12(0.050 g), selenium (0.045 g)/100 ml Glycine has been tried duringdevelopment: 0.01% to 1% w/v.

Glycine conforming to EP monograph has been tested.

Following concentrations of glycine have been tested:

-   -   5% w/v: a dark clear solution with particle is obtained    -   10% w/v: a dark clear solution without particle is obtained    -   15% w/v: a dark clear solution without particle is obtained

Physical stability study is performed on 5 and 15% w/v glycine (withvitamin B12) solutions at ambient temperature:

-   -   T1 week: dark clear solution with particle    -   T2 weeks: dark clear solution with particle    -   T4 weeks: dark clear solution with particle

Use of Antioxidants

Use of antioxidant is evaluated, with butylhydroxyanisole BHA andbutylhydroxytoluene BHT. Other antioxidants have used: propyl gallate,ascorbic acid, rongalite, sodium metabisulphate.

Following antioxidant trials have been tested:

-   -   1% w/v BHA and BHT: incomplete solubilisation

As a result of low solubility of BHA and BHT in water, processsolubilisation of antioxidant(s) must be reviewed.

CONCLUSION

In conclusion, satisfactory results with butaphosphan solutions wereobtained with different concentrations.

Results after 4 weeks of physical stability study showed dark clearsolution.

These formulations were selected to go to next phase.

3.2. Phase 2: Laboratory Batches

According results obtained with vitamin B12 and butaphosphan solutions,laboratory batches are performed.

(a) Formula 1

Ingredients Quantity per formula in mg Manganese (Manganese Carboante)10.0 Zinc (Zinc Oxide) 60.0 Copper (Copper Sulphate Pentahydrate) 15.0Selenite (Sodium Selenite Anhydrous) 5.0 Butaphosphan 100.0 Vitamin B121.5

Excipients benzyl alcohol, edetic acid, sodium hydroxide and water forinjection

Process

(b) Formula 2

Ingredients Quantity per formula in mg Manganese (Manganese Carbonate)4.0 Zinc (Zinc Oxide) 24.0 Copper (Copper Sulphate Pentahydrate) 6.0Selenite (Sodium Selenite Anhydrous) 2.0 Butaphosphan 100.0 Vitamin B121.5

Excipients benzyl alcohol, edetic acid, sodium hydroxide and water forinjection

Process

4. Phase 2: Stability Studies

A stability study at 25° C./60% RH and 40° C./75% RH was performed onformulations candidate during 3 months.

The aim of this study was to screen the formulations stable underambient and accelerated conditions.

A visual inspection will be checked at different periods in order tocontrol the absence of precipitation in the formulations: at 5° C., 25°C./60% RH and 40° C./75% RH: T0, T1, T2 and T3 months.

Assays of Vitamin B12 were evaluated: follow up on stability at 25°C./60% RH and 40° C./75% RH: T0, T3 and T6 months.

SUMMARY

The method of preparing a trace element solution in accordance with theinvention thus enables the production of an injectable solutioncomprising an adequate trace mineral concentration and Vitamin B12 sothat a 5 to 10 millilitre injection can make a significant impact on thetrace mineral status of the animal and an injection is provided at arate of between 1 ml per 50 kg bodyweight (BW) and 1 ml per 100 kg BW,i.e. a practically applicable injectable supplement and a product thatcan improve the trace mineral status of an animal is provided. This isimportant as livestock producers will only inject livestock if a realbenefit can be demonstrated. The subcutaneous injection is the preferredroute to minimize tissue damage, but intra-muscular injection can alsobe used.

1. A trace element solution, which comprises at least the followingmetals: (a) zinc; (b) manganese; (c) selenium; and (d) copper; and whichcomprises Vitamin B12.
 2. A solution as claimed in claim 1, in which:(a) the ratio of zinc to manganese is at least 2:1; (b) the ratio ofzinc to manganese is at least 4:1; (c) the ratio of zinc to copper is atleast 2:1 or 4:1; and/or (d) the ratio of zinc to selenium is at least4:1 or 12:1.
 3. A solution as claimed in claim 1, which comprises thefollowing concentrations: (a) at least 24 mg/ml zinc; (b) at least 4mg/ml manganese; (c) at least 2 mg/ml selenium; (d) at least 6 mg/mlcopper; and (e) at least 0.6 mg/ml Vitamin B12.
 4. A solution as claimedin claim 1, in which the concentration of the metals is at least 36mg/ml.
 5. A solution as claimed in claim 1, which comprises thefollowing concentrations: (a) at least 60 mg/ml zinc; (b) at least 10mg/ml manganese; (c) at least 5 mg/ml selenium; (d) at least 15 mg/mlcopper; and (e) at least 1.5 mg/ml Vitamin B12.
 6. A solution as claimedin claim 1, in which the concentration of the metals is at least 90mg/ml.
 7. A solution as claimed in claim 1, which comprises chromiumand/or iodine.
 8. A solution as claimed in claim 1, which comprisesbutaphosphan to stabilize the Vitamin B12.
 9. A solution as claimed inclaim 5, in which the inclusion of butaphosphan enables the Vitamin B12to remain more stable.
 10. A solution as claimed in claim 1, which is aninjectable trace element solution.
 11. A solution as claimed in claim 1,which is visually stable. 12-13. (canceled)
 14. A method of providingtrace elements to animals comprising administering the solution ofclaim
 1. 15. A solution as claimed in claim 2, which comprises thefollowing concentrations: (a) at least 24 mg/ml zinc; (b) at least 4mg/ml manganese; (c) at least 2 mg/ml selenium; (d) at least 6 mg/mlcopper; and (e) at least 0.6 mg/ml Vitamin B12.
 16. A solution asclaimed in claim 2, in which the concentration of the metals is at least36 mg/ml.
 17. A solution as claimed in claim 2, in which theconcentration of the metals is at least 90 mg/ml
 18. A solution asclaimed in claim 2, which comprises butaphosphan to stabilize theVitamin B12
 19. A method of providing trace elements to animalscomprising administering the solution of claim
 2. 20. A method ofproviding trace elements to animals comprising administering thesolution of claim
 5. 21. A method of providing trace elements to animalscomprising administering the solution of claim
 6. 22. A method ofproviding trace elements to animals comprising administering thesolution of claim 9.